Increased microvascular permeability causes pulmonary edema in acute lung injury, adult respiratory distress syndrome (ARDS), and multisystem organ failure. Disruption of endothelial cell-cell contacts is a possible cause of increased microvascular permeability. Identification of intracellular mechanisms which regulate changes in vascular permeability may lead to therapeutic strategies for controlling tissue damage due to sepsis or other causes of acute lung injury. Our preliminary results indicate that overexpression of protein kinase Cdelta (PKCDELTA) protects against thrombin-induced increases in endothelial monolayer permeability. The hypothesis of this proposal is that PKCDelta blunts changes in endothelial monolayer permeability by modulating the organization of cytoskeleton junctional complexes. Endothelial cells (EC) normally form a tightly adherent monolayer. During an inflammatory or thrombogenic response endothelial monolayer permeability increases with the formation of interendothelial cell gaps as a result of both EC contraction and disruption of endothelial cell-cell interactions. EC contraction occurs by intracellular calcium mobilization, Rho GTPase activation, myosin light chain phosphorylation, actin microfilament reorganization, and focal adhesion complex (FA) formation. Disruption of endothelial cell-cell junctions occurs concomitantly with the redistribution of the adherens junction (AJ) protein complexes at the points of intercellular gap formation. The intracellular mechanisms by which edemagenic agents promote EC contraction and FA formation and the disassembly of the AJ complexes are not completely understood. A number of inflammatory and thrombogenic agents activate PKC, increasing monolayer endothelial permeability. The overall objective of this proposal is to determine the mechanism by which PKCDELTA modulates endothelial barrier function. Using microvascular EC which stably overexpress PKCDELTA and vector control, we will determine: I) whether diminished endothelial barrier function is due to the stabilization and association of AJ or FA with PKCDELTA; II) whether delta PKCDELTA blunts endothelial barrier function by modulating EC retraction at the level of actin stress fiber formation and/or function; and III) whether PKCDELTA reduces endothelial barrier function by altering MAPK signaltransduction pathway. The elucidation of the molecular mechanisms by which PKCDELTA decreases endothelial monolayer permeability may lead to the development of therapeutic agents which are protective against endothelial barrier dysfunction.